3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method.
The modification of tyrosine residues in proteins to 3-nitrotyrosine by peroxynitrite or other potential nitrating agents has been detected in biological systems that are subject to oxidative stress. A convenient semi-quantitative method has been developed to assay nitrated proteins in biological fluids and homogenates using a competitive ELISA developed in our laboratory. This assay selectivity detected 3-nitro-l-tyrosine residues in a variety of peroxynitrite-treated proteins (BSA, human serum albumin (HSA), alpha1-antiprotease inhibitor, pepsinogen and fibrinogen) and also in a nitrated peptide, but had a low affinity for free 3-nitro-L-tyrosine and 3-chloro-L-tyrosine. The IC50 values for the inhibition of antibody binding by different nitrated proteins were in the range 5-100 nM, suggesting that the antibody discriminated between nitrotyrosine residues in different environments. The presence of nitrotyrosine in plasma proteins was detected by Western blot analysis and quantified by the ELISA. A concentration of 0. 12+/-0.01 microM nitro-BSA equivalents was measured in the proteins of normal plasma which was increased in peroxynitrite-treated plasma and was elevated in inflammatory conditions. HSA and low-density lipoprotein (LDL) isolated from plasma contained 0.085+/-0.04 and 0. 03+/-0.006 nmol nitro-BSA equivalents/mg protein, respectively. Comparison of the level of nitration in peroxynitrite-treated HSA and LDL in the presence and absence of plasma indicates that nitration and presumably oxidation is inhibited by plasma antioxidants. The presence of nitrotyrosine in LDL is consistent with previous reports implicating peroxynitrite in the oxidative modification of lipoproteins and the presence of a low concentration of oxidized LDL in the blood.
Khan
J
J
Department of Pharmacology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, U.K.
Brennand
D M
DM
Bradley
N
N
Gao
B
B
Bruckdorfer
R
R
Jacobs
M
M
Brennan
D M
DM
D000328:Adult; D001667:Binding, Competitive; D001798:Blood Proteins / Q000737:chemistry*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D008077:Lipoproteins, LDL / Q000097:blood; D012709:Serum Albumin / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D001798:Blood Proteins; D008077:Lipoproteins, LDL; D012709:Serum Albumin; C002744:3-nitrotyrosine; D014443:Tyrosine